A number of therapeutic proteins, including clotting factors, immunoglobulin (IVIG), and albumin, are purified from human plasma by companies like Grifols, Baxter, and CSL. It's important to test for parvovirus B19 and HAV because they are non-enveloped viruses, making these viruses resistant to inactivation during the purification (fractionation) process. Relatively low levels of B19 are allowed to exist is a plasma fraction (current regulations require less than 10,000 IU in a manufacturing pool which can contain 4,000 to 5,000 individual plasma units). Rather than taking the risk of assembling a large manufacturing pool and then finding out that it's contaminated with B19, plasma fractionators typically screen smaller pools to identify individual plasma units that contain high titers of B19. There are currently no regulations related to HAV but testing is generally performed because doing so has become an industry standard.
Human parvovirus (genus Erythrovirus) is a blood borne, non-enveloped virus that has a single-stranded DNA (ssDNA) genome of about 5.5 kb (Shade et al., 1986, J. Virol. 58(3): 921-936, Brown et al., 1997, Ann. Rev. Med. 48: 59-67). Individual virions contain one copy of either the plus or minus strand of the genome, represented in approximately equal numbers. The ssDNA genome has inverted terminal repeats that form 5′ and 3′ hairpins of about 350 nt, which are essential for viral replication. The genome includes two open reading frames on the plus strand, which code for structural proteins (VP1 and VP2) and non-structural protein (NS1).
At one time it was believed that human parvovirus was highly conserved at less than 2% genetic diversity. More recently, though, it has been discovered that a human Erythrovirus isolate, originally termed V9, has a greater than 11% divergence in genome sequence compared to B19, with the most striking DNA dissimilarity at >20%, observed within the p6 promoter region. The V9 isolate was determined to have a clinical presence of greater than 11%, as well. Now the human Erythrovirus group is divided into three distinct virus genotypes: genotype 1 (B19), genotype 2 (A6- and LaLi-like), and genotype 3 (V9-like). (Servant et al., 2002, J. Virol. 76(18): 9124-34; Ekman et al., 2007, J. Virol. 81(13): 6927-35). Servant et al., refer to genotype 1 as viruses corresponding to parvovirus B19 and refer to genotypes 2 and 3 as viruses corresponding to parvovirus V9-related. Ekman et al., refer to genotypes 1-3 as all corresponding to parvovirus B19. For convenience herein, genotypes 1, 2 and 3 are referred to as parvovirus genotypes 1, 2 and 3 or human parvovirus genotypes 1, 2 and 3. Nucleic acid detection assays that do not accurately detect all parvovirus genotypes result in many plasma pools remaining contaminated with human parvovirus. Thus, there is a need for a nucleic acid test that detects human parvovirus genotypes 1, 2 and 3.
Infection with human parvovirus can occur via respiratory transmission or through infected blood or blood products. Infected individuals may exhibit no symptoms, or have erythema infectiosum symptoms that include mild flu-like symptoms, rash (“fifth disease”), temporary arthritis-like joint pain (arthropathy), aplastic crisis in patients with hemolytic anemias, persistent parvovirus infection and loss of about 10% of early pregnancies due to fetal death. Thus, the failure to detect parvovirus in a pooled plasma sample or for diagnosis of infection has serious consequences.
Further, there is a need that detection assays provide a detection sensitivity that allows for detection of low titers of virus, as may occur early in an infection or in diluted or pooled samples. Parvovirus nucleic acid detection assays that can detect an appropriate level of contamination may facilitate removal of infected donated units from the blood supply or contaminated lots of pooled plasma before use.
Many immunodiagnostic methods detect anti-parvovirus antibodies (IgM or IgG) present in an individual's serum or plasma (e.g., see PCT Nos. WO 96/09391 by Wolf et al. and WO 96/27799 by Hedman et al.). These methods have limitations in detecting recent or current infections because they rely on detecting the body's response to the infectious agent. The rapid rise in viremia following infection results in high levels of parvovirus in an individual's blood without corresponding detectable levels of anti-parvovirus antibodies (See, e.g., U.S. Pat. No. 7,094,541 to Brentano et al at Example 4). Thus, immunological-based detection assays are susceptible to false negative results. Furthermore, viremia is often quickly cleared, yet a person may remain antibody-positive in the absence of these infective particles, thusly leading to false positive results. As many as 90% of adults are seropositive for parvovirus, making accurate immunological detection of recent or current infections difficult. Other similar assays detect the presence of parvovirus by detecting the virus or empty viral capsid bound to a purified cellular receptor (U.S. Pat. No. 5,449,608 to Young et al.), and these immuno-based assays experience similar problems.
DNA hybridization and amplification methods have also been used to detect human parvovirus, though these tests are generally directed to the detection of genotype 1 only. Yet, U.S. and European regulatory bodies have promulgated standards specifying that plasma pools used for manufacturing anti-D immunoglobulin and other plasma derivatives can contain no more than 10,000 IU/ml (10 IU/microliter in Europe) of any human parvovirus. As discussed above, therapeutic plasma pools and diagnostic tests need similarly to reliably identify human parvovirus types 1, 2 and 3. Thus, there is a need in the art for compositions, kits and methods useful in the in vitro nucleic acid detection of human parvovirus types 1, 2 and 3.
Hepatitis A virus (HAV) is the causative agent of one form of hepatitis that may produce symptoms that include fever, fatigue, nausea, abdominal pain, diarrhea, loss of appetite, and jaundice over less than two months. Of those infected with HAV, about 10% to 15% have a prolonged or relapsing symptoms over a six to nine months following infection. Immunity to HAV, based on the individual's production of anti-HAV immunoglobulin G (IgG), follows both symptomatic and asymptomatic infections.
Although the incidence of HAV infections has dramatically decreased in parts of the world in which vaccination for HAV has been widely used since the late 1990's, epidemics of HAV infections (>700 cases per 100,000 population, and for children who live in areas with high rates of hepatitis A the rate increases to >20 cases per 100,000 population) may occur in non-immune populations where poor sanitary conditions exist, even temporarily, e.g. following an earthquake. Transmission may also result from contact with HAV-contaminated serum or blood products. Even in the USA, every year about 100 persons die from acute liver failure due to hepatitis A (death rate of about 0.015%). Even in nonfatal hepatitis A cases, substantial costs are associated with HAV infections, including those that result from patient hospitalization, outpatient visits, and lost work days.
HAV is a 27-nm RNA virus (a picornavirus) that contains a plus-sense single-stranded RNA genome of about 7.5 kb. The virus replicates in the liver, is excreted in bile, and is shed in feces (e.g., up to 108 virus per ml) during the acute phase of infection. The incubation period is usually two to six weeks before symptoms appear. A single serotype of HAV has been found worldwide. Diagnosis of hepatitis A cannot be differentiated from other types of viral hepatitis by symptoms or other clinical features (e.g., elevated serum aminotransferase levels). Typically, hepatitis A diagnosis is confirmed by serological testing that provides positive results for the presence of anti-HAV immunoglobulins (Ig). Anti-HAV IgM is generally present five to ten days before the onset of symptoms and is undetectable in most patients by six months later, whereas anti-HAV IgG appears early during infection and remains detectable for the individual's lifetime. HAV RNA can be detected in the blood and stool of most persons during the acute phase of infection by using nucleic acid testing methods, e.g., amplification by the polymerase chain reaction (PCR), and nucleic acid sequencing has been used to identify the genetic relatedness of HAV following community-wide infections (Dato et al., Morbidity Mortality Wkly. Rpt., 2003, 52(47): 1155-57; LaPorte et al., Morbidity Mortality Wkly. Rpt., 2003, 52(24): 565-67). These methods, however, are not generally used for diagnostic purposes.
Therefore, there exists a need to accurately detect the presence of HAV in the biological samples and environmental samples. There is also a need to detect the presence of HAV contamination in products that may be used in medical treatment (e.g., blood or serum for transfusions, or factors derived from human blood or serum). There is also a need to detect the presence of HAV in potentially contaminated materials, such as water or food, to prevent community-wide outbreaks or epidemics resulting from consumption of contaminated materials.
The inventions disclosed herein respond to those needs by describing oligonucleotide sequences that are used in nucleic acid testing methods to detect the presence of HAV nucleic acid (HAV RNA or sequences derived therefrom, e.g., cDNA). This application also describes nucleic acid testing methods that detect the presence of HAV RNA present in a sample.